Gram Staining Procedure and Concepts

These flashcards cover the fundamental concepts and detailed procedures related to Gram staining in microbiology.

Who invented the Gram Stain?

Christian Gram in 1884.

What is the main purpose of the Gram Stain?

To differentiate bacteria into Gram positive and Gram negative.

What are the two groups of bacteria identified by the Gram Stain?

Gram positive and Gram negative bacteria.

What is the primary stain used in the Gram Stain procedure?

Crystal Violet.

What role does Gram’s Iodine play in the Gram Staining process?

It acts as a mordant and binds to Crystal Violet.

What happens to Gram positive bacteria when alcohol is applied?

They remain purple due to the thick peptidoglycan layer.

What happens to Gram negative bacteria when alcohol is applied?

They become clear due to the thin peptidoglycan layer.

What is the function of Safranin in the Gram Stain process?

It serves as a counterstain for Gram negative bacteria.

What color do Gram positive bacteria appear after the counterstaining step?

Purple.

What color do Gram negative bacteria appear after the counterstaining step?

Red/Pink.

What is the purpose of heat fixing in Gram staining?

To adhere bacterial cells to the slide.

What is the first step in performing a Gram Stain?

Add a loopful of water to the slides for each bacterium.

What are the materials needed for the Gram Staining exercise?

Microscope slides, nutrient agar plates, water blanks, bacterial cultures.

What happens if the alcohol is left on the slide for too long?

A Gram positive bacterium may appear Gram negative.

What type of bacteria is Bacillus subtilis classified as?

BSL1.

What type of bacteria is Staphylococcus aureus classified as?

BSL2.

What should you label on the nutrient agar plate before streaking?

Your name, bacteria name, and initials.

How many drops of Ethyl Alcohol are used in the Gram staining procedure?

8-10 drops.

How long should Crystal Violet stain the cultures?

1 minute.

How long should the Gram’s Iodine stain the cultures?

1 minute.

How long should Safranin stain the cultures?

1 minute.

What is the significance of peptidoglycan in Gram stains?

It determines whether bacteria are Gram positive or negative.

What should be done after adding Safranin to the cultures?

Gently rinse with water.

What happens if the smear is too thick?

A Gram negative bacterium may mistakenly appear Gram positive.

What do you use to blot the slide dry?

Kimwipe.

Which object should you start with when observing under the microscope?

The 4x objective.

What does the term 'mordant' refer to in the context of Gram staining?

A substance that binds to a dye and makes it less soluble.

How should you apply the Ethyl Alcohol during the Gram stain?

Holding the slide at a downward angle.

What color is the Gram stain before the application of Safranin?

Purple for all bacteria.

During which step do you need to make sure to avoid heat fixing?

When preparing a negative stain or capsule stain.

What is the morphology step in the Gram staining process?

Noting each bacterium's appearance and Gram reaction.

How long should you heat fix the slides?

Pass through the flame 3 times.

What do you need to do with your loop when transferring cultures?

Sterilize it after each quadrant use.

Why is it important to allow the samples to air dry before heat fixing?

To prevent boiling the bacteria and damaging them.

What is meant by 'aseptically add' in a microbiology lab?

To add without contaminating the sample.

What are the consequences of damaging bacterial cell walls during the Gram stain process?

It may lead to incorrect Gram reactions.

How long should you let the alcohol decolorizer sit on the slide?

5-10 seconds.

Why is it important to rinse the slide gently with water?

To avoid washing away the stains.

What should you do if working with soil bacteria according to the procedure?

No need to add water, use them directly from liquid culture.

How many quadrants are created on the nutrient agar plate?

Four quadrants.

What is the purpose of providing thin and thick smears?

To obtain a sufficient view of bacterial morphology.

What is an important precaution while working with bacteria?

Maintain aseptic technique.

Which type of culture should one of the students use on the agar plate?

A different bacterial culture from their partner.

What should happen immediately after adding Crystal Violet?

Allow staining for 1 minute.

When is the only time you should not heat fix a slide?

When preparing a negative stain or a capsule stain.

What should be noted during observation of the samples?

The morphology and Gram reaction of each organism.

What is the purpose of the nutrient agar plate in this context?

To cultivate bacteria for observation.